Proteases are critical factors enabling many protozoa and helminths to parasitize and survive in both mammalian and insect hosts. The biological properties and differential expression of related groups of zinc metalloproteases in the African trypanosome Trypanosoma brucei will be studied using the techniques of traditional gene deletion, RNA interference (RNAi), and overexpression for selected experiments. Three groups of metalloproteases, called TbMSP-A, TbMSP-B and TbMSP-C, each has about 33% amino acid identity with the major surface protease (MSP, formerly called GP63) of Leishmania, and share a common zinc binding motif. The mRNAs of all three gene families occur in bloodstream trypanosomes, whereas only the mRNA of TbMSP-B is expressed in procyclic trypanosomes. Their C-terminal amino acid sequences suggest that TbMSP-A and -B are likely associated with a cellular membrane via a glycosylphosphatidylinositol (GPI) anchor, whereas TbMSP-C may be either located in the cytoplasm or secreted. We hypothesize that those TbMSPs predicted on the basis of their sequences to traffic to the trypanosome surface, i.e., TbMSP-A and TbMSP-B, serve important functions in facilitating survival of trypanosomes in the host environment. Our preliminary data lead us to hypothesize that TbMSP-B is a surface protease under translational control in bloodstream cells, which participates in shedding of the variant surface glycoprotein (VSG) during differentiation from bloodstream to procyclic cells. Further evidence leads us to hypothesize that TbMSP-A serves a distinct proteolytic function that is not required in procyclic cells, although the exact nature of this function has yet to be identified. In this application we propose to use the techniques of immunolocalization, sub-cellular fractionation, metabolic labeling, protease characterization, and gene mutagenesis to identify the sub-cellular locations of the TbMSP-A and TbMSP-B families, to determine their biological functions, and to elucidate the molecular mechanisms regulating their differential expression.